作者衷心感谢王培acknowledge博士提供的缺氧室仪器，以及Alice Lui女士的技术支持。

所有涉及动物的手续均严格按照NIH"实验动物护理和使用指南"进行，并经香港大学李嘉诚医学院动物实验动物教学与研究委员会批准。获得知情同意后，从健康供体的髂嵴获得人骨髓样品。香港大学机构审查委员会批准议案。 1.大鼠MSC培养物的制备<ol><li> 从股骨收获MSC <ol><li>在使用前，将所有的夹层工具（ 即精细剪切剪刀，钝头切割剪刀和齿形钳子）在180°C下高压灭菌至少2小时。 </li><li>制备包含补充有15％胎牛血清（FBS）和青霉素/链霉素（P / S，1％v / v）的最小必需培养基-α修饰（αMEM）的MSC生长培养基）。 </li><li>用戊巴比妥过量（240mg / kg体重，腹膜内）牺牲年轻的雄性Sprague Dawley大鼠（体重200-250g）。 注意:来自不同大鼠的骨髓样品应分开处理。 </li><li>将牺牲的动物置于仰卧位。用70％乙醇彻底清洁他们的腹部和下肢。 </li><li>使用精细的解剖剪刀和镊子去除内侧大腿上的皮肤和皮下组织。沿周向除去大腿肌肉直到股骨暴露。继续向近端和远端，直到看到膝盖和髋关节。通过髋关节和膝关节脱位股骨，使用钝头切割剪刀。 注意:在这个阶段不要横断股骨以露出骨髓腔。将完整的股骨转移到层流组织培养罩以进一步处理。 </li><li>使用钝头切割剪刀穿过干to端穿过股骨的远端和近端。 </li><li>将70μm的细胞过滤器放在50mL锥形管上。将含有磷酸盐缓冲盐水（PBS，10 mM Na 2 HPO 4 ，pH 7.4）的21 G，10 mL注射器插入裸露的股管，并通过反复冲洗将骨髓内容物冲洗成锥形管。 注意:使用约20 mL PBS冲洗每个股骨。如果被冲洗的内容物的颜色保持血染和混浊，则可以使用较大的体积。 </li><li>通过480×g离心5分钟收集细胞。丢弃上清液。将细胞沉淀重悬于10mL的MSC生长培养基中。将细胞置于10厘米的组织培养皿上。将组织培养皿放入细胞培养箱（37℃，5％CO 2 ）中。将电镀的初始日期记录为第0天。 注意:在涉及离心的所有步骤中，设置最大减速度的制动器。 </li></ol></li><li> 建立和扩建MSC殖民地 注意:本协议r组织培养上的精液塑性粘附作为从骨髓腔内选择MSC的手段11 。允许骨髓含量粘附到组织培养塑料上2天。 <ol><li>在第2天，用10mL PBS冲洗培养板三次以去除非贴壁细胞。冲洗后用10mL的生长培养基替换PBS。用PBS洗涤细胞，每3天补充MSC生长培养基。 注意:MSC殖民地在第6-7天应该可见（ 图2A ）。 </li><li>在第10天通过除去生长培养基并用PBS冲洗细胞，使细胞通过细胞。加入1.5 mL重组酶切细胞解离试剂，37℃孵育5 min。加入3 mL MSC生长培养基中和反应。通过以250×g离心5分钟收集分离的细胞。 </li><li>在PBS中适当稀释后，使用血细胞计数器量化沉淀中的细胞。 </li><li>将种子传代细胞以40,000个细胞/ cm 2的密度转移到MSC生长培养基中的10cm培养板中。 注意:大鼠MSC在传代后2天内应达到80-90％汇合（ 图2B ）。细胞可以如步骤1.2.2所述传代达8代。 MSC可以通过免疫细胞化学和它们的三线分化能力来表征（ 图3 ）。通道3和8之间只有MSC培养物经受随后的缺氧预处理和神经祖细胞富集。更大通道数的MSC采用扁平形态（ 图2C ），不产生足够数量的神经祖细胞。这些文化应该被丢弃。 </li></ol></li></ol> 2.人类BMSC培养物的制备<ol><li>用9 mL MSC生长培养基稀释1 mL人骨髓抽吸液，细胞在10厘米的组织培养皿上。将培养物保持在细胞培养箱（37℃，5％CO 2 ）中。 </li><li> 2天后取出培养基，用10mL PBS轻轻冲洗培养液三次以去除非贴壁细胞。最后冲洗后，取出PBS，并用10 mL的MSC生长培养基更换。在PBS冲洗后每3天培养一次，补充生长培养基。 注意:第6-7天，MSC菌落应该可见。殖民地的数量可能会在受试者之间变化。 </li><li>在第10天通过细胞，如步骤1.2.2所述。在PBS中适当稀释后，使用血细胞计数器量化沉淀中的细胞。将传代细胞以40,000个细胞/ cm 2的密度接种到MSC生长培养基中的10cm培养板上。 注意:人类MSC（ 图2D ）显示与大鼠MSC相似的形态，同样应在传代后2天内达到80-90％汇合。他们应该是chara通过免疫细胞化学和它们的三线分化能力进行调整12 。与大鼠MSC一样，在第3代和第8代之间的人类MSC接受后续的缺氧预处理和神经祖细胞富集。 </li></ol>缺氧预处理<ol><li>拆开环形夹具后，拆除缺氧室部件（ 即底座，盖子，托盘），并用70％乙醇清洁各个部件。将腔室部件置于层流组织培养罩内，在UV光下灭菌15分钟。 </li><li>在缺氧预处理之前，取出培养基，并用10mL PBS冲洗大鼠和人MSC培养物（第1和第2部分）。用10mL补充有25mM HEPES的MSC生长培养基替换PBS。 注意:培养在10厘米盘子上的MSC在进行缺氧预处理前应达到80-90％的汇合度。 </li><li>放cul在缺氧室内的菜肴。重新组装腔室部件并拧紧环夹。将99％N 2 /1％O 2的气体混合物以10升/分钟的流速冲洗到室中5分钟。 </li><li>密封缺氧室的连接端，确保没有气体泄漏。将细胞培养箱（37℃，5％CO 2 ）放置室16小时。 </li><li>完成缺氧预处理后，从室中取出培养物，为后续的神经祖细胞富集培养做准备。 </li></ol>神经祖细胞丰富文化<ol><li>制备由补充有B27（2％v / v），碱性成纤维细胞生长因子（bFGF，20ng / mL），表皮生长因子（EGF，EGF）的Dulbecco修饰的Eagle培养基/ Ham的营养混合物F12（DMEM / F12）组成的神经祖细胞培养基20ng / mL）和P / S（1％v / v）。 </li><li>分离缺氧预处理的大鼠/人类MSC，如步骤1.2.2所述。通过以250×g离心5分钟收集分离的细胞。使用血细胞计数器在PBS中适当稀释后，定量细胞沉淀。 </li><li>将细胞重悬于神经祖细胞培养基中，并以6,000个细胞/ cm 2的密度将其种子置于低附着的6孔板上。将培养物置于细胞培养箱（37℃，5％CO 2 ）中12天。每3天补充75％的神经祖细胞。 注意:在第6-7天应该观察到大小的非粘附细胞簇。在第10-12天，可以观察到直径≥100μm的神经球（ 图4 ）。与在正常氧条件下培养的MSC相比，低氧预处理的MSC应该产生更多的神经球。 </li><li>通过将其吸入10 mL移液管并将其转移到15 mL锥形管中，在第12天收集神经球。离心神经球250 xg 5分钟。 注意:神经球可以特征在第12天用于神经祖细胞标志物，如巢蛋白和GFAP 7 。 </li></ol>通过与DRG神经元的共培养生成命运致命的雪旺氏细胞<ol><li> 制备纯化的大鼠DRG神经元 <ol><li>在使用前，将所有的夹层工具（ 即，夹层剪刀，镊子，两个显微切割钳和显微解剖剪刀）高压灭菌至少2小时。 </li><li>在4℃下，将具有聚-D-赖氨酸（PDL，10μg/ mL PBS）的6孔组织培养板过夜。取出PDL，每孔用1.5 mL PBS冲洗。 </li><li>继续在37℃下用层粘连蛋白（10μg/ mL，PBS）包被2小时。每孔用1.5 mL PBS冲洗平板。 </li><li>制备DRG神经元维持培养基，其中包括补充有B27（2％v / v），L-谷氨酰胺（1％v / v），神经生长因子（NGF，20ng / mL）和P / S（1 ％v / v）。 </li><li>制备DRG神经元纯化培养基，由补充B27（2％v / v），L-谷氨酰胺（1％），NGF（20ng / mL），P / S（1％），氟脱氧尿苷（FDU，10 μg/ mL）和尿苷（10μg/ mL）。 </li><li>通过戊巴比妥过量（240mg / kg体重，腹膜内）在妊娠14-15日怀孕大鼠。 </li><li>将牺牲的动物置于仰卧位。用70％乙醇彻底清洁腹部。 </li><li>使用精细的解剖剪刀和镊子纵向切割动物的下腹壁。使用解剖剪刀识别并清除子宫。切割子宫壁暴露和提取胚胎。将胚胎转移到装有PBS的无菌10cm培养皿中。将培养皿放在冰上。 </li><li>将用于解剖的胚胎转移到装有PBS的无菌10cm培养皿（室温）中并将其置于解剖显微镜下。让胚胎处于俯卧位。 不E:发白的脊髓和附着的DRG可以通过其半透明的皮肤在胚胎的背部看到。 </li><li>沿脊髓的两侧插入微分解钳，并使用钝性剥离开始将脊髓与周围的软组织分开。使用显微解剖镊子沿着颈部开口和尾巴切割没有动物的脊髓。在电线的腹侧进行进一步的钝性剥离，以将其从周围的软组织中释放出来。 </li><li>使用微分解钳去除残留的软组织，在脱离脊髓的背侧。 注意:在这个阶段，只有脊髓，神经根和附着的DRG应该保留。 </li><li>使用显微解剖钳从其连接的神经根分离个体DRG。使用连接到1 mL尖端的移液器笔将DRG转移到含有PBS的1.5 mL无菌离心管中。 注意:对于每个1.5 mL管，最多为可以容纳100个DRG。 </li><li>以250 xg的速度将DRG离心5分钟，并重新悬浮于重组酶切细胞解离试剂（每管200μL）。孵育（37℃，5％CO 2 ）10分钟。以250 xg离心DRG 5分钟，取出上清液，重悬于DRG神经元维持培养基中。通过使用200μL移液管尖端轻轻研磨分离沉淀。在适当稀释后使用血细胞计数器量化沉淀中的细胞。 </li><li>将细胞以5,000个细胞/ cm 2的密度种植在每孔1.5mL的DRG神经元维持培养基中的PDL /层粘连蛋白包被的6孔板中。培养两天后，取出DRG神经元维持培养基，用PBS冲洗，更换为DRG神经元纯化培养基。 注意:对于每个纯化周期，用纯化培养基处理DRG培养物2天，然后在维持培养基中孵育1天。 3-4次纯化循环后，预期所有内源性神经胶质的卵圆形7 。这应该需要大约14天。纯化的培养物对神经元标记TUJ1测试为阳性，并且S100β表达缺失（ 图5 ）。 </li></ol></li><li> 雪旺细胞样细胞的产生 <ol><li>制备补充有β-非调白蛋白（100ng / mL），bFGF（10ng / ml），血小板衍生生长因子（PDGF-AA，5ng / mL），FBS（10％）和P / S（1％v / v）。 </li><li>将第4部分制备的神经球以每孔5-10微米的密度，每孔5-10毫升的神经胶质细胞诱导培养基在PDL /层粘连蛋白包被的6孔板中制备。用PBS清洗细胞后，每2天更换一次胶质细胞诱导培养基。 注意:接种的神经球的细胞被认为在第2天向外迁移。到第7天，迁移细胞具有锥形外观，并应证明施万细胞的免疫阳性标志物p75神经营养因子受体（p75）和S100β7。这些细胞被称为SCLC。 </li></ol></li><li> SCLCs与DRG神经元的共培养 <ol><li>以1:1体积与体积比制备由DRG神经元维持培养基（步骤5.1.4）和胶质诱导培养基（步骤5.2.1）组成的共培养培养基。 </li><li>准备由补充有FBS（5％），β-非调白蛋白（10ng / mL）和P / S（1％v / v）的DMEM / F12组成的施万细胞维持培养基。 </li><li>从第7天SCLC中取出培养基，用PBS冲洗，并将其与0.5mL /孔的重组酶切细胞解离试剂在37℃孵育5分钟。将SCLC重悬于共培养培养基中。 </li><li>在适当稀释后用血细胞计数器量化细胞。 </li><li>将SCLC以1,000个细胞/ cm 2的密度接种到纯化的DRG神经元培养物上。保持共培养14天，每2天更换一次。 注意:在共培养期间，SCLC获得了成熟的施万细胞典型的纺锤状形态（ 图6 ）。这些细胞在生长因子撤出后仍然保持其表型，并且能够在体外和体内髓鞘髓鞘7,10。施万细胞标志物（ 即 p75和S100β）的阳性应通过免疫荧光进行监测。 </li><li>共同培养完成后，通过命运提出的施万细胞，如步骤1.2.2所述。每孔使用0.5 mL离解试剂。在适当稀释后用血细胞计数器量化细胞。 </li><li>以10,000个细胞/ cm 2的密度重悬在施万细胞维持培养基中的命运承诺的雪旺氏细胞。将种子细胞分成PDL /层粘连蛋白包被的6孔板进行免疫荧光。 注意:共培养不可避免地含有已经采用成纤维细胞的MSC细胞命运7 。在共同培养完成后，这些细胞与命运致命的施万细胞一起传播。位于该成纤维细胞基质顶部的雪旺氏细胞可以容易地分离，并且在PBS（4℃）的"冷喷"中冲洗后进一步扩大，如其他地方所述。命运承诺的施万细胞可以在维持培养基中扩增1个月。 </li></ol></li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mesenchymal+stem+cells:+characterization,+differentiation,+and+application+in+cell+and+gene+therapy.">Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy.</a> J Cell Mol Med. 8 (3), 301-316 (2004).</li><li>Jirsova, K., Sodaar, P., Mandys, V., Bar, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+jet:+a+method+to+obtain+pure+Schwann+cell+cultures+without+the+need+for+cytotoxic,+apoptosis-inducing+drug+treatment.">Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment.</a> J. Neurosci. Methods. 78 (1-2), 133-137 (1997).</li></ol>

本手稿的所有作者均无披露声明。

在通过缺氧预处理和神经球培养富集神经祖细胞之前，必须保持MSC的"干性"。根据我们的经验，可以通过其细长的成纤维细胞样形态可靠地鉴定多能MSC。相比之下，采用更扁平，四边形形态，具有突出的细胞骨架应力纤维的MSC不容易采用神经细胞命运，应该被丢弃。一般来说，我们不使用通道数大于8的MSC。为了保持它们的干性，在达到100％汇合之前及时通过MSC是至关重要的。相反，保持MSC在一个?...

神经祖细胞及其衍生物的移植显示出创伤神经损伤1,2和神经变性3,4之后的治疗策略的希望。在临床应用之前，必须确保:i）获取和扩大自体来源的干/祖细胞的方法，以及ii）将其引导到相关的成熟细胞类型的手段3 。我们对细胞治疗脊髓损伤的兴趣导致我们从成人组织中寻找一个健壮的自体细胞来源的神经祖细胞。 MSCs的亚群源自神经嵴，并且容易从骨髓腔进入。这些细胞是可以产生神经元和神经胶质的神经祖细胞5 。脑缺血的动物模型表明，缺氧促进了唾液<html>脑内神经祖细胞的多发性和多能性6 。这是利用缺氧预处理作为扩展骨髓来源的神经祖细胞的手段的基础。 将施万细胞移植到损伤的脊髓中促进再生2 。 SCLC可以通过补充Gliogenic因子（ 即， β-Heregulin，bFGF和PDGF-AA）从MSC产生，但表现出表型不稳定性。退出生长因子后，它们恢复成为成纤维细胞样表型7 。由于异常分化和致癌的风险，细胞移植中的表型不稳定性是不合需要的。由于施旺细胞前体与胚胎周围神经8内的轴突束相关，我们被引导与纯化的胚胎DRG神经元共培养SCLC 7 ，ass ="xref"&gt; 9。结果成熟的雪旺氏细胞是命运决定的，并在体外证明功能7,9和体内 10 。 我们用于富集MSC的神经祖细胞的方案是简单和有效的，导致后续测定的细胞数量增加。通过共培养平台推导命运致命的雪旺氏细胞允许研究胶质细胞分化和产生用于潜在临床应用的稳定和功能的施万细胞。

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">&#945;MEM</td>
			<td style="width:140px;">Sigmaaldrich</td>
			<td style="width:101px;">M4526</td>
			<td style="width:329px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12</td>
			<td>Thermofisher scientific</td>
			<td>12400-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal medium</td>
			<td>Thermofisher scientific</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Biosera</td>
			<td>FB-1280/500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27</td>
			<td>Thermofisher scientific</td>
			<td>17504-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal growth factor (EGF)</td>
			<td>Thermofisher scientific</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic fibroblast growth factor (bFGF)&#160;</td>
			<td>Peprotech</td>
			<td>100-18B/100UG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nerve growth factor (NGF)&#160;</td>
			<td>Millipore</td>
			<td>NC011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet-derived growth factor-AA (PDGF-AA)</td>
			<td>Peprotech</td>
			<td>100-13A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heregulin beta-3, EGF domain (&#946;-Her)</td>
			<td>Millipore</td>
			<td>01-201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Uridine</td>
			<td>Sigmaaldrich</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Fluro-2&#39; - deoxyuridine (FDU)</td>
			<td>Sigmaaldrich</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-D-lysine (PDL)</td>
			<td>Sigmaaldrich</td>
			<td>P7886-1G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin</td>
			<td>Thermofisher scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMAX</td>
			<td>Thermofisher scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin / streptomycin (P/S)</td>
			<td>Thermofisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE Express</td>
			<td>Thermofisher Scientific</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 cm plate for adherent culture</td>
			<td>TPP</td>
			<td>93100</td>
			<td>Used for selection of MSCs by tissue culture adherence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well plate for adherent culture</td>
			<td>TPP</td>
			<td>92006</td>
			<td>Used for expansion of MSCs following passaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraLow 6-well plate for non-adherent culture</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Used for neural progenitor enrichment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human CD90(Thy-1)</td>
			<td>BD Biosciences</td>
			<td>555593</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human CD73</td>
			<td>BD Biosciences</td>
			<td>550256</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human/rat STRO-1</td>
			<td>R&#38;D Systems</td>
			<td>MAB1038</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human nestin</td>
			<td>R&#38;D Systems</td>
			<td>MAB1259</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>555480</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD90(Thy-1)</td>
			<td>BD Biosciences</td>
			<td>554895</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD73</td>
			<td>BD Biosciences</td>
			<td>551123</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat nestin</td>
			<td>BD Biosciences</td>
			<td>MAB1259</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD45</td>
			<td>BD Biosciences</td>
			<td>554875</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-S100&#946;</td>
			<td>Dako</td>
			<td>Z031101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-p75</td>
			<td>Millipore</td>
			<td>MAB5386</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-GFAP</td>
			<td>Sigmaaldrich</td>
			<td>G3893</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Class III-beta tubulin (Tuj-1)</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Human nuclei</td>
			<td>Millipore</td>
			<td>MAB1281</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hypoxia chamber</td>
			<td>Billups-Rothenberg</td>
			<td>MIC-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES buffer</td>
			<td>Sigmaaldrich</td>
			<td>H4034-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

hypoxic preconditioning of marrow-derived progenitor cells as a source for the generation of mature schwann cells

该手稿描述了一种从骨髓基质细胞（MSC）群体中丰富神经祖细胞并随后将其引导至成熟的施万细胞命运的手段。我们将大鼠和人类MSC接受短暂的缺氧条件（1％氧气16小时），随后在含有表皮生长因子（EGF）/碱性成纤维细胞生长因子（bFGF）补充的低附着底物上扩增为神经球。将神经球接种到聚-D-赖氨酸/层粘连蛋白包被的组织培养塑料上，并在含有β-Heregulin，bFGF和血小板衍生生长因子（PDGF）的胶质细胞混合物中培养以产生施旺细胞样细胞（SCLC）。 SCLC通过共同培养2周，以E14-15怀孕的Sprague Dawley大鼠获得的纯化的背根神经节（DRG）神经元为指导。成熟的雪旺氏细胞表现出S100β/ p75表达的持续性，并可形成髓鞘细胞。以这种方式生成的细胞具有潜力脊髓损伤后自体细胞移植中的应用，以及疾病建模。

我们的协议关键阶段的概述如图1所示 。总之，通过依靠组织培养塑料选择大鼠和人类MSC。扩张的MSC被缺氧预处理，然后经历神经球形成条件。电镀神经球，使其分化成SCLC。 SCLC与纯化的DRG神经元共培养以产生命运赋予的施旺细胞。 培养的大鼠和人类MSC的形态如图2所示 。显示出与?...

Watch this Scientific Journal Video about Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells at JoVE.com

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

具有神经电位的骨髓基质细胞（MSC）存在于骨髓内。我们的方案通过缺氧预处理丰富了这种细胞群，之后引导他们成为成熟的雪旺氏细胞。

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

hypoxic-preconditioning-marrow-derived-progenitor-cells-as-source-for

Research

JoVE Journal

Developmental Biology

7.2K 次观看.  The University of Hong Kong. 具有神经电位的骨髓基质细胞（MSC）存在于骨髓内。我们的方案通过缺氧预处理丰富了这种细胞群，之后引导他们成为成熟的雪旺氏细胞。 

视频: Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

小鼠心脏场特定心脏祖细胞的体外生成

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...